Numerous nucleic acid amplification techniques have been devised, including strand displacement cascade amplification (SDCA)(referred to herein as exponential rolling circle amplification (ERCA)) and rolling circle amplification (RCA)(U.S. Pat. No. 5,854,033; PCT Application No. WO 97/19193; Lizardi et al., Nature Genetics 19(3):225-232 (1998)); multiple displacement amplification (MDA)(PCT Application WO 99/18241); strand displacement amplification (SDA)(Walker et al., Nucleic Acids Research 20:1691-1696 (1992), Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396 (1992)); polymerase chain reaction (PCR) and other exponential amplification techniques involving thermal cycling, self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), and amplification with Qβ replicase (Birkenmeyer and Mushahwar, J. Virological Methods 35:117-126 (1991); Landegren, Trends Genetics 9:199-202 (1993)); and various linear amplification techniques involving thermal cycling such as cycle sequencing (Craxton et al., Methods Companion Methods in Enzymology 3:20-26 (1991)).
Rolling Circle Amplification (RCA) driven by DNA polymerase can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (Lizardi et al., Nature Genet. 19: 225-232 (1998); U.S. Pat. Nos. 5,854,033 and 6,143,495; PCT Application No. WO 97/19193). If a single primer is used, RCA generates in a few minutes a linear chain of hundreds or thousands of tandemly-linked DNA copies of a target that is covalently linked to that target. Generation of a linear amplification product permits both spatial resolution and accurate quantitation of a target. DNA generated by RCA can be labeled with fluorescent oligonucleotide tags that hybridize at multiple sites in the tandem DNA sequences. RCA can be used with fluorophore combinations designed for multiparametric color coding (PCT Application No. WO 97/19193), thereby markedly increasing the number of targets that can be analyzed simultaneously. RCA technologies can be used in solution, in situ and in microarrays. In solid phase formats, detection and quantitation can be achieved at the level of single molecules (Lizardi et al., 1998). Ligation-mediated Rolling Circle Amplification (LM-RCA) involves circularization of a probe molecule hybridized to a target sequence and subsequent rolling circle amplification of the circular probe (U.S. Pat. Nos. 5,854,033 and 6,143,495; PCT Application No. WO 97/19193). Very high yields of amplified products can be obtained with exponential (or cascade) rolling circle amplification (U.S. Pat. Nos. 5,854,033 and 6,143,495; PCT Application No. WO 97/19193) and multiply-primed rolling circle amplification (Dean et al., Genome Research 11:1095-1099 (2001)).
Change in cellular function is related to the changes in the expression pattern of different proteins. Thus, valuable information about cellular function is often obtained by comparing the expression of specific proteins between two stages of cell growth, or function (Zhu et al., “Global analysis of protein activities using proteins chips.” Science 293(5537): 2101-5 (2001); Schweitzer and Kingsmore, “Measuring proteins on microarrays.” Curr. Opin. Biotechnol. 13(1): 14-9 (2002); Schweitzer et al., “Multiplexed protein profiling on microarrays by rolling-circle amplification.” Nat. Biotechnol. 20(4): 359-65 (2002)). This endeavor is referred to as expression profiling. Since protein expression profiling studies are mostly dependent on the availability of protein specific antibodies, many of the gene expression-profiling studies are, therefore, carried out at the level of mRNA expression (Colantuoni et al., “Gene expression profiling in postmortem Rett Syndrome brain: differential gene expression and patient classification.” Neurobiol. Dis. 8(5): 847-65 (2001); Colantuoni et al., “High throughput analysis of gene expression in the human brain.” J. Neurosci. Res. 59(1): 1-10 (2000)). Thus, important information about the disease process or treatment regime can be obtained by evaluating the relative expression of a gene in two different cellular environments (for example, cancer vs. normal tissue, or drug treated vs. non-treated states). Current high throughput expression profiling studies often are carried out on DNA micro-arrays. Gene specific DNA probes are anchored on to glass slides and fluorescent cDNA or amplified cDNA (from the RNA sample) is hybridized to these anchored probes and detected. Utilization of DNA microarrays is expected to provide advances in identifying genetic profile of human diseases by the acquired ability to screen thousands of individual genes that may be expressed differentially between two samples (Colantuoni et al. (2000); Colantuoni et al. (2001); Nallur et al., “Signal amplification by rolling circle amplification on DNA microarrays.” Nucleic Acids Res. 29(23): E118 (2001)). Typically, these assays require 5-100 μg of total RNA for a single analysis. Millions of cells are, therefore, required to obtain sufficient RNA sample for expression profiling experiments with unamplified RNA samples. Often only a few cells that were acquired by aspiration needle biopsies, rare population subsets isolated by cell sorting, laser capture micro dissections or micro manipulated single cells, are available for expression profiling experiments. In such instances only small quantities of RNA becomes available for expression profiling experiments. Therefore, many high throughput expression-profiling studies need to be carried out using amplified cDNA samples. However, most current cDNA amplification technologies have shortcomings that result in amplification bias. Therefore, there is a need for a method for reliable, universal, unbiased transcript amplification.